Deborah Dempsey

Rapid Generation and Flow Cytometric Analysis of Stable GFP-Expressing Cells

Expression of green fluorescent protein (GFP) represents a unique method for the fluorescent labeling of viable mammalian cells, with many potential applications. The studies detailed in this report examine the detection of GFP expression in murine cells using flow cytometry. Direct comparison of NIH 3T3 cells transiently expressing GFP or GFPS65T, a mutant form of GFP, showed that GFPS65T fluorescence (using 488 nm excitation) was detected more readily than fluorescence from wildtype GFP. Efficient generation of cell lines that stably expression GFPS65T was achieved using a plasmid vector that encoded a hygromycin phosphotransferase/GFPS65T fusion protein. Flow cytometric detection of NIH 3T3 cells expressing this fusion protein was improved using a 510/20 band pass filter instead of the standard filter setup for fluorescein detection. Additionally, staining the surface of these cells with phycoerythrin, RED 670, or allophycocyanin did not interfere with the detection of GFPS65T fluorescence, indicating that multiparameter analyses using GFPS65T fluorescence are possible. Importantly, we also observed that GFPS65T expression could be detected in NIH 3T3, BW5147, or freshly cultured Thy1lo CD3- murine bone marrow cells transduced with a retroviral vector encoding the fusion protein, suggesting that the potential applications of this system may be quite broad.

Dual-color flow cytometric detection of fluorescent proteins using single-laser (488-nm) excitation.

The ability to analyze independently the expression of multiple reporter gene constructs within single cells is a potentially powerful application of flow cytometry. In this paper, we explore the simultaneous detection of two variants of the reporter molecule, green fluorescent protein (GFP) that both fluoresce when excited with 488-nm light. One of these, enhanced GFP (EGFP) (excitation max. 490 nm; > 90% efficiency at 488 nm), has been widely used for studies that involve flow cytometric detection of reporter gene expression. As a partner for EGFP, we employed a recently described variant termed enhanced yellow fluorescent protein (EYFP) (excitation max. 513 nm; approximately 35% efficiency at 488 nm). Using 488-nm excitation, EYFP fluorescence could be readily detected following expression of the gene in murine fibroblasts and this signal was comparable in intensity to that obtained from EGFP. Importantly, we describe an optical filter configuration that permits the fluorescence signals from both proteins to be distinguished by flow cytometry, despite their similar emission maxima. This filter configuration employed a 510/20-nm bandpass filter for EGFP detection, a 550/30-nm bandpass filter for EYFP detection, and a 525-nm short-pass dichroic mirror to separate the two signals. With these filters, expression of either reporter protein could be detected, alone or in combination, within a mixed population of cells over a broad range of signal intensities.

Expression of Intracellular IFN-γ in HSV-1-Specific CD8+ T Cells Identifies Distinct Responding Subpopulations During the Primary Response to Infection

Cutaneous infection in the footpads of C57BL/6 mice with HSV-1 results in an accumulation of activated (CD44high CD25+) CD8+ T cells within the draining popliteal lymph node (PLN). These studies were undertaken to evaluate the frequency and phenotype of the CD8+ T cell population within the PLN, recognizing the single immunodominant HSV-1 epitope derived from the viral envelope glycoprotein, glycoprotein B (gB), using an intracellular IFN-γ-staining assay. It revealed that ∼6% of the CD8+ T cells were specific for the gB epitope. Phenotypic analysis of the IFN-γ-producing gB-specific CD8+ T cells generated in the PLN during the course of the acute infection expressed the CD44high CD25+ phenotype on days 3–5 postinfection. Surprisingly, IFN-γ-producing CD8+ T cells expressed the CD44high CD25− phenotype on days 5–8 postinfection, in contrast to expectations for a CD8+ effector T cell. IFN-γ-producing CD25− CD8+ T cells were detected in the PLN on day 21 postinfection, long after infectious virus had been cleared. Throughout the response, the spleen was found to be the major reservoir of gB-specific CD8+ T cells, even during the peak of the response. In contrast to the gB-specific CD8+ T cell population within the PLN, the entire gB-specific CD8+ T cell population within the spleen was CD25−. Collectively, these results suggest the generation of subpopulations of virus-specific CD8+ T cells, distinguished by the expression of CD25, during the acute phase of the primary response to a localized viral infection.

Characterization of the progeny of pre-T cells maintained in Vitro by IL-3: Expression of the IL-2 receptor and CD3 during thymic development

We have recently described a bone marrow culture system which is able to maintain, for at least 2 weeks, cells which have the capacity to repopulate the thymus of irradiated recipient mice (pre-T cells). Because this culture system depends upon the addition of an exogenous growth factor (IL-3) which may potentially influence the differentiation of the cultured pre-T cells, it is important to determine whether or not the progeny of cultured marrow cells are able to develop within the thymus in a kinetically normal fashion. Here we report the results of an analysis of the progeny of those cultured progenitor cells at 2, 3, and 4 weeks following intrathymic transfer. The passage of cultured donor-derived cells through critical early (expression of the IL-2 receptor) and late (expression of high levels of CD3) intrathymic events was assessed in these studies and compared with the pattern observed in the progeny of fresh bone marrow cells. The results of these studies showed that the progeny of cultured pre-T cells were able to develop expression of the IL-2 receptor and CD3 surface antigen during their residency within the thymus. In addition, both the timing and levels of expression of these surface markers were virtually identical on the progeny of fresh and cultured pre-T cells. These data suggest that cultured pre-T cells are not dramatically altered by their passage in vitro and are able to give rise to normally developing thymocytes upon in vivo transfer.

Pre-thymic transcription of TCR genes by adult murine bone marrow cells.

In the adult mouse, the earliest thymocytes are derived from bone marrow-resident T lymphocyte precursor (pre-T) cells that immigrate to the thymus. There they undergo maturation through a series of developmental steps that include rearrangement and expression of the TCR genes, positive and negative selection, and functional maturation. Although these intrathymic processes have been extensively characterized, little is known about the T cell-specific events that take place in the bone marrow microenvironment. Of particular interest are the events surrounding transcription and rearrangement of the various TCR chains that are required for functional TCR expression. We have previously reported the transcription of incompletely rearranged TCR beta genes in pre-T cell-containing fractions of adult bone marrow. Here we demonstrate that the TCR gamma chain genes are also transcriptionally active in these cells. Like the TCR beta transcripts, TCR gamma transcripts are sterile, originating from unrearranged gamma loci. Interestingly, both RAG-1 and RAG-2 transcripts were also detected in this cell fraction, suggesting that sterile TCR transcription might be dependent upon the presence of a functional recombinase system. However, both C beta and C gamma sterile transcripts could be detected from the same bone marrow cell population isolated from RAG-1 gene deficient mice. Therefore, the expression of TCR genes can initiate at the earliest stages of T cell development, prior to exposure to the thymic microenvironment, and a functional recombinase system is not required for the production of these sterile TCR transcripts.

Characterization of the progeny of pre-T cells maintained in vitro by IL-3: Appearance in the periphery and Vβ utilization in vivo

We have recently demonstrated that bone marrow-resident cells, which are able to repopulate the thymus of irradiated recipient mice (pre-T cells), can be maintained in vitro for at least 2 weeks in the presence of exogenous IL-3. Because this marrow culture system can be applied to the study of early T cell differentiation, it is important to ascertain the extent to which in vitro culture of the pre-T cells might alter the T cell progeny which can develop from them. In previous work, we showed that the progeny of cultured pre-T cells appeared to develop in a kinetically normal fashion within the thymus of recipients and that the acquisition of key developmental markers (IL-2R and CD3) was identical in the progeny of fresh and cultured pre-T cells. Here, we report the results of experiments carried out to characterize the progeny of cultured pre-T cells which were found in the peripheral lymphoid tissues several weeks following intrathymic transfer to irradiated recipients. We found no remarkable differences between the progeny of cultured or fresh marrow cells with respect to the timing of their appearance in the periphery nor their expression of CD4 or CD8. By studying the patterns of utilization of five different V beta gene products by the T cells derived from fresh or cultured bone marrow, we were able to test the susceptibility of both sets of progeny to both positive and negative selection pressures during their in vivo maturation. These experiments established that the progeny of cultured marrow cells were equally susceptible to TCR repertoire selection, as were the progeny of fresh bone marrow cells, and that the process of in vitro growth did not alter the potential TCR repertoire of the pre-T cells.